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First published online 17 November 2009
doi: 10.1242/jcs.054791
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Research Article |
Np63
through the p38 pathway
1 Department of Physiology, College of Medicine, Chang Gung University, Kweishan, Taoyuan 333, Taiwan
2 Section of Blood Products & IVDs, Drug Biology Division, Bureau of Food and Drug Analysis, Department of Health, Taiwan
* Author for correspondence (jkc508{at}mail.cgu.edu.tw)
Accepted 26 September 2009
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Summary |
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Np63
expression by KGF is mediated via the p38 pathway. The effect of KGF on limbal epithelial outgrowth was significantly reduced when endogenous Np63 was suppressed, suggesting that KGF-induced limbal epithelial outgrowth is dependent on the expression of Np63. Our findings strongly suggest that limbal keratocytes regulate limbal epithelial cell growth and differentiation through a KGF paracrine loop, with Np63 expression as one of the downstream targets.
Key words: Limbus, Cornea, Stem cell, Epithelium, KGF, Np63
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Introduction |
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). Experimental evidence has shown that slow-cycling and label-retaining cells are preferentially present in the epithelial basal layer of the limbus (Cotsarelis et al., 1989), the transition zone between cornea and conjunctiva. It has also been shown that a corneal differentiation marker, cytokeratin-3, was absent from the limbal basal layer, suggesting a more primitive nature for this cell layer (Schermer et al., 1986). Damages to limbal epithelial stem cells might lead to chronic inflammation, fibrous tissue ingrowth and invasion of vascularized conjunctiva into the cornea, leading to functional blindness (Tseng, 1989; Chen and Tseng, 1990; Huang and Tseng, 1991).
The epithelial-mesenchymal interactions through humoral factors have important roles in maintenance of corneal integrity and function, and in wound healing. The cellular factors involved in such crosstalk include transforming growth factor- (TGF-), TGF-β, interleukin-1 (IL-1) (Williams et al., 1992; Forge et al., 1993), platelet-derived growth factor (PDGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and others. These factors are thought to be secreted by epithelial cells and/or the underlying keratocytes (Thoft and Friend, 1983; Wilson et al., 1994b; Li and Tseng, 1995). In addition to humoral factors, extracellular matrix components and cell-membrane-associated molecules are also believed to be involved.
KGF and HGF are two prototypic fibroblast-derived paracrine mitogenes of epithelial cells. KGF belongs to the fibroblast growth factor family (FGF7), and has been found to be secreted by the stromal cells of a variety of tissues, including cornea, skin, prostate and mammary gland (Wilson et al., 1993; Sotozono et al., 1994; Werner et al., 1994a; Tang and Gilchrest, 1996; Liu et al., 1998). KGF binds with high affinity to only the KGF receptor (KGFR), an alternative splicing variant of the FGF receptor 2 (FGFR2), which is expressed exclusively on the epithelial cells (Wilson et al., 1993). KGF is a well-established mitogen for keratinocytes; however, it has also been shown to promote early differentiation and inhibit terminal differentiation of the cultured keratinocytes (Marchese et al., 1990). HGF was originally identified in liver cells, and is also known as scatter factor because of its ability to stimulate cell migration (Gherardi et al., 1993). HGF acts through binding to a specific receptor, c-Met, and has been reported to promote keratinocyte proliferation (Weidner et al., 1990; Furlong et al., 1991; Montesano et al., 1991; Johnson et al., 1993) and stimulate keratinocyte metalloproteinase production in response to skin injury, migration and proliferation (Dunsmore et al., 1996).
The transcription factor p63 exhibits high levels of sequence and structural homology to p53, and is regarded as a member of the p53 family (Yang et al., 1998). As a result of alternative promoter usage, two major isoforms of p63, TAp63 and Np63 are generated. Proteins with a transactivation domain at the N-terminus are named TAp63, and those lacking this domain are named Np63 (Yang et al., 1998). It has now been well established that during embryonic development, programmed expression of p63 is crucial for the development and maturation of various epithelia. p63-knockout mice have been shown to exhibit severe developmental deficiencies in limbs, hair follicles, teeth and mammary glands, suggesting that p63 has a crucial role in the development of stratified epithelia (Mills et al., 1999). Recent studies suggested that TAp63 is required for the initiation of the epithelial stratification program, whereas Np63 is required for the epidermal differentiation (Koster et al., 2004; Koster and Roop, 2004; McKeon, 2004). In human skin keratinocyte organotypic cultures, knockdown of p63 resulted in defective stratification and impaired expression of epithelial differentiation markers (Truong et al., 2006). Initially, p63 was thought to be a specific limbal stem cell marker in human cornea (Pellegrini et al., 2001); however, later studies have shown that its expression is not confined to the limbal basal layer in rat cornea (from age 1 day to 3 months) (Hsueh et al., 2004). RT-PCR examination of the spatial distribution of p63 isoforms in human ocular surface epithelia revealed that Np63 is the most dominant isoform expressed (Kawasaki et al., 2006). Moreover, Np63 has been shown to be more specific than other isoforms as a marker for limbal epithelial stem cells. The preferential expression of Np63 in activated limbal basal layer suggests that it is involved in maintenance of the proliferative potential of limbal stem cells (Di Iorio et al., 2005). P63 has been reported to promote keratinocyte proliferation by inhibiting the transcription of p21 (Westfall et al., 2003), and has been found to be overexpressed in many squamous carcinomas, suggesting that it is involved in normal development and carcinogenesis (Boldrup et al., 2005; Rocco and Ellisen, 2006; de Oliveira et al., 2007). In a wounded cornea, limbal stem cells are activated to produce daughter cells to repair the damaged area, and these new descendent cells are strongly p63 positive. After corneal repair is completed, p63 expression is again confined mostly to the basal cells of the limbal epithelium, strongly indicating that p63 is involved in the regulation of cornea stem cell proliferation and differentiation. In a previous study, we found that blockage of TAp63 expression promotes limbal epithelial cell differentiation, and that blockage of Np63 expression inhibits cell proliferation (Wang et al., 2005).
In this study, we show that KGF is a better mitogen than HGF in promoting epithelial outgrowth from limbal explants in amniotic membrane (AM)-based low-serum culture. Immunohistochemical examination of the cell sheet of the limbal outgrowth obtained from HGF and KGF treated cultures showed no obvious difference in the expression of differentiation markers, such as keratin-3 and keratin-14. By contrast, the expression level of p63 was greatly enhanced in KGF-treated, but not in HGF-treated cells. Furthermore, KGF induced Np63 expression through the p38 pathway. Knockdown of Np63 expression led to attenuation of the KGF-stimulated limbal epithelial outgrowth. Interestingly, transfected Np63 alone exhibited no stimulatory effect on limbal epithelial outgrowth. The results suggest that the upregulation of Np63 is necessary, but not sufficient, for the promotion of limbal epithelial outgrowth by KGF.
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; Liu et al., 1998). During corneal wound repair, mRNA levels of HGF, KGF and their receptors have been found to be upregulated (Werner et al., 1992; Werner et al., 1994b; Wilson et al., 1999). To assess the possible effect of HGF and KGF on limbal epithelial cell growth in a normal cornea, we probed the expression levels of HGFR and KGFR in the corneal and limbal epithelium by immunohistochemistry. We found that HGFR is strongly expressed in suprabasal to superficial layers of the corneal and limbal epithelia, whereas KGFR is strongly expressed in the basal layer of the limbus, but is weakly expressed throughout the corneal epithelium and the suprabasal layers of the limbal epithelium (Fig. 1A).
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To better understand how HGF and KGF promote limbal epithelial cell proliferation, the possible effect of HGF and KGF on cell cycle progression was analyzed by flow cytometry. As shown in Fig. 1F, the G0-G1 population of the control culture was estimated to be 86.45%; upon HGF and KGF treatment, the G0-G1 population was reduced to 81.2% and 80.3%, respectively. In conjunction with a reduced G0-G1 cell population, the S-phase population increased from 3.3% to 7.2% and 9.7%, respectively, upon HGF and KGF treatment.
KGF promotes limbal epithelial outgrowth through the induction of Np63
AM-based limbal explant culture has been shown to be a suitable culture system for the growth and maintenance of limbal explant outgrowth in a phenotypic characteristic similar to that of the in vivo limbal tissue (Wang et al., 2003). This culture system is therefore also suitable for studying the effect of growth factors on limbal epithelial cell growth and differentiation. Limbal explants were cultured on AM and stimulated separately with HGF (80 ng/ml), KGF (20 ng/ml) or HGF+KGF for 10 days. On day 10, culture dishes were stained with trypan blue and photographed (Fig. 2A). As shown in Fig. 2B, the size of limbal epithelial outgrowth grown in the presence of KGF (2.3±0.3 cm2) or HGF+KGF (2.1±0.2 cm2) was significantly larger than that grown in the presence of HGF (1.3±0.4 cm2) and control (<0.1 cm2) cultures.
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Our previous results showed that blockage of TAp63 and Np63 expression inhibits limbal epithelial outgrowth on AM (Wang et al., 2005). P63 was once regarded as a specific marker for corneal stem cells, and Np63 was shown to maintain epithelial homeostasis by regulating cell proliferation and differentiation (Pellegrini et al., 2001; Koster et al., 2004; Truong et al., 2006). We therefore, sought to examine whether the aforementioned growth factors affected the expression of p63 in the cells of the limbal outgrowth. We found that p63 was highly expressed in the basal cells of the epithelial outgrowth in cells treated with KGF and HGF+KGF, but not in HGF-treated cells (Fig. 3D-F).
Limbal epithelial cell protein lysates were analyzed by western blotting for the presence of Np63, and blots were compared with that of a Np63-expressing cell line, HaCaT (Barbieri et al., 2003; Matheny et al., 2003). The results suggest that the predominant isoform expressed is Np63. To confirm the western blot result, RT-PCR reactions were performed using 11 specific primer pairs that are able to identify every TAp63 and Np63 isoforms (supplementary material Table S1). As shown in Fig. 4, transcripts for the TA domain, β-specific and 
-specific regions were not detected. By contrast, Np domain, -specific and Np63-specific region were detected. The RT-PCR result indicated that Np63 is the predominant isoform species produced by the limbal epithelial sheet outgrowth.
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Np63 (Fig. 5B). In the dose range used, higher outgrowth stimulation by KGF was correlated with higher Np63 protein expression.
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Np63 by KGF is mediated via the p38 pathwayNp63 was dose-dependently stimulated by KGF (Fig. 6A).
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Np63 expression (Fig. 6C,D). These results indicate that the induction of Np63 expression by KGF is mediated via activation of the p38 pathway.
Knockdown of p63 attenuates KGF-induced limbal epithelial outgrowth
To determine whether KGF-promoted limbal epithelial outgrowth is mediated through induction of Np63 expression, specific siRNA was used to knock down p63 expression. The epithelial cells of the limbal explant culture were transfected with control or p63 siRNA (both at 80 µM) and cultured in the presence or absence of KGF for 10 days. After this, the cells from KGF-treated cultures were harvested and probed for Np63 expression by western blotting. As shown in Fig. 7A, the Np63 protein level was effectively suppressed by p63 siRNA transfection. The epithelial outgrowth in KGF-treated cultures was significantly reduced from 1.3±0.1 cm2 to 0.9±0.2 cm2 upon p63 siRNA transfection (Fig. 7B). In cultures without KGF, the epithelial outgrowth was minimal and was not obviously affected by siRNA transfection (Fig. 7B,C). Taken together, these data demonstrate that the expression of Np63 is necessary for KGF-induced limbal epithelial outgrowth.
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Expression of Np63 alone is not sufficient to promote limbal epithelial outgrowth
Since the blockage of Np63 expression suppresses KGF-induced limbal epithelial outgrowth, we therefore sought to investigate whether overexpression of Np63 in limbal epithelial cells is sufficient to promote limbal epithelial outgrowth. For this purpose, a human Np63 adenoviral expression vector was constructed. The primary limbal epithelial cells were transduced with Ad-Np63 (with a V5-tag) or Ad-GFP. Transfection with Ad-GFP served as a control for the estimation of transfection efficiency and any effect on cell proliferation. The transfection efficiency was estimated to be around 80%, and the transfection exhibited little effect on cell growth (data not shown). As shown in Fig. 8A, the Ad-Np63-transfected cells expressed a V5-tagged Np63 protein band, which was not present in the Ad-GFP-transfected cells. Ad-Np63-transfected cells exhibited a faster growth rate than Ad-GFP transfected cells cultured under the same conditions (Fig. 8B). To see whether transfected Np63 also promoted epithelial outgrowth from the limbal explants, we transduced limbal explant cultures with Ad-Np63 or Ad-GFP and further cultured the explants for 10 days in the presence or absence of KGF (0-20 ng/ml). However, Np63 transfection did not significantly promote limbal epithelial outgrowth from the explants with or without KGF treatment (Fig. 8C). In our culture system, the endogenous Np63 expression was relatively high compared with the ectopically expressed Np63 (Fig. 8D). This might partially explain the lack of effect of the ectopically expressed Np63 in promoting limbal epithelial outgrowth. The failure of ectopic Np63 to promote limbal epithelial outgrowth suggests a saturation effect with elevated endogenous levels of Np63, or that regulation of limbal epithelial proliferation and/or migration by KGF involves a more complicated mechanism.
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; Meller and Tseng, 2000; Tseng et al., 2002; Ti et al., 2004). AM-based limbal explant culture has proved to be a good culture system to expand limbal epithelial cells to form limbal-like epithelia equivalents (Wang et al., 2003). The AM-based limbal epithelial equivalents have been successfully used to reconstruct the surface of limbal-deficient cornea, implying that the primitive corneal epithelial cells or possibly corneal stem cells are probably preserved and expanded upon AM-based limbal explant culture (Tsai et al., 2000). Because of its successful clinical application, it is therefore of cell biological and clinical significance to study the regulation of the growth and differentiation of the limbal epithelial cells cultured on AM.
Numerous studies have shown that limbal stem cell deficiency as a result of chemical burns, thermal burns or Stevens-Johnson syndrome might lead to chronic inflammation of the corneal surface and eventually functional blindness. Therefore, understanding the regulation of limbal stem cell proliferation and differentiation is of great ophthalmological significance. Earlier reports by others showed that the expression levels of HGF and KGF receptor mRNAs are markedly upregulated in mouse corneal epithelium after scrape injury (Wilson et al., 1999). KGF and HGF have been shown to promote corneal wound healing through the activation of the PI3K pathway (Chandrasekher et al., 2001). In addition, KGF has also been shown to be a potent inducer of epithelial cell differentiation (Marchese et al., 1997). In the present study, we compared the effect of KGF and HGF on the proliferation and differentiation of limbal epithelial sheet grown on AM.
To see whether there might be differential responses of the limbal epithelial cells to HGF and KGF, we first examined the expression pattern of HGFR and KGFR in corneal-limbal epithelium. HGFR was found to be strongly expressed in suprabasal to superficial layers of the corneal and limbal epithelia, whereas the KGFR was mainly expressed in the basal layer of the limbal epithelium. The results suggest that the target areas of HGF and KGF on the ocular surface epithelium are different. In addition, KGF promoted outgrowth of limbal epithelia better than HGF, when compared on a molar basis (1 µM). Since the epithelial outgrowth from limbal explant is inversely correlated with the rapidity of cell differentiation, we therefore examined the expression patterns of keratin-3 and keratin-14 in the epithelial outgrowth. We found that the expression patterns of these two keratin proteins were not obviously different between HGF- and KGF-treated cultures, even though it has been reported that KGF and HGF regulate keratinocyte differentiation in different manners (Wilson et al., 1994b).
The expression of p63 is confined to the basal cells of the limbal epithelium, and has been suggested to be a marker of low keratinocyte differentiation (Pellegrini et al., 2001; Koster et al., 2004; Wang et al., 2005). In the present study, we therefore sought to examine whether HGF and KGF influence p63 expression in different manners. Interestingly, we found that Np63 was strongly expressed in the cells of KGF-treated limbal explant cultures, but not in those treated with HGF. Since HGF and KGF did not appear to alter the differentiation status of the cells as judged from expression of keratin-3 and keratin-14, we therefore suggest that the mitogenic effect of KGF is mediated through Np63 expression. Our assumption is supported by the observation that knockdown of Np63 expression by siRNA attenuated the growth-promoting effect of KGF, and further suggested that the expression of Np63 is necessary for KGF mitogenicity for limbal epithelial cells. As shown in Fig. 3, it is obvious that the number of cell layers in the HGF-treated limbal epithelial sheet was lower than that of groups treated with KGF and KGF+HGF. We attribute this discrepancy to the lack of Np63 expression in the HGF-treated explants. Consistent with this, Np63 expression has been shown to be necessary for normal multilayered epithelial development and stratification (Truong et al., 2006).
Increased p38 phosphorylation has been shown to correlate with increased Sp1 activity (D'Addario et al., 2006). A putative Sp1-binding site is present in the Np63 promoter region, and Sp1 has been shown to associate with the Np63 promoter region in mouse keratinocytes (Romano et al., 2006).
To see whether the expression of Np63 alone is sufficient to promote limbal epithelial cell proliferation, Np63 vector was transfected into limbal epithelial cells and the cell proliferation was compared with that of GFP-transfected cells in both monolayer and explant cultures. Np63 transfection increased limbal epithelial cell proliferation in monolayer cultures; however, it did not promote cell outgrowth in explant cultures, with or without KGF treatment.
It has been shown that KGFR is upregulated in human keratinocytes during Ca2+-induced differentiation, and the KGF signaling pathway might control the proliferative and differentiation from basal to suprabasal layer of the human skin epithelium (Marchese et al., 1997). Capone and colleagues (Capone et al., 2000) also showed that Ca2+-induced differentiation of the human keratinocyte cell line HaCaT resulted in the upregulation of KGFR. Thus, KGF-KGFR signaling appears to have dual roles in promoting keratinocyte proliferation and regulating differentiation. In limbal explant culture, the expression of Np63 has been shown to be necessary for epithelial outgrowth (Wang et al., 2005), and in the present study, KGF-promoted outgrowth is also shown to be dependent on Np63 expression.
A recent report by Candi and co-workers (Candi et al., 2007) showed that in p63–/– mice, the KGFR expression was greatly reduced, and was restored to normal upon reintroduction of Np63. They also showed that overexpression of Np63 significantly increased KGFR mRNA levels in the Saos-2 cell line. These observations suggest a role of p63 in KGFR expression. These, together with a previous report showing that KGF is specifically expressed in limbal keratocytes, support the assumption that KGF-KGFR and the downstream expression of Np63 are implicated in the regulation of limbal keratinocyte proliferation.
Collectively, our data support a role for KGF-induced Np63 expression in the stimulation of limbal epithelial outgrowth on AM. KGF is a better stimulator than HGF, and the effect is mediated via the p38 pathway. We conclude that the induction of Np63 has a necessary, but not sufficient role in KGF-induced limbal epithelial sheet outgrowth on AM.
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Np63 polyclonal antibody was purchased from BioLegend (San Diego, CA). ERK1/2-P (Thr202/Tyr204), JNK-P (Thr183/Tyr185), phosphorylated p38 MAP kinase (Thr180/Tyr182) were purchased from Cell Signaling (Beverly, MA). Sp1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay kit was purchased from Roche Applied Science (Mannheim, Germany). All plastic cell culture wares were from Corning Incorporated Life Sciences (Acton, MA).
Preparation of denuded human amniotic membrane (AM)
Human AM was obtained from Chang Gung Memorial Hospital (Linko, Taiwan) with proper informed consent, and were processed as described (Koizumi et al., 2001). Briefly, AM was washed three times in 100 ml of 1x phosphate buffer saline (PBS) containing 50 µg/ml penicillin, 50 µg/ml streptomycin, and 25 ng/ml gentamicin. AM was then serially washed once with 0.5, 1, 1.5 mM DMSO in PBS, and was finally incubated with 2.4 U/ml dispase II at 37°C for 20 minutes to loosen the epithelial layer. The epithelial cells were removed by gentle scraping with cell scraper (Nunc, Napierville, IL), and the denuded AM was then placed on six-well culture plates with the basement-membrane side up and incubated in a humidified incubator (37°C, 5% CO2 in air) overnight before use.
Limbal epithelial cell culture
Rabbit limbal epithelial cells were isolated and cultivated as described (Pellegrini et al., 1999). Briefly, limbal tissue was treated with trypsin (0.05% and 0.01% EDTA) at 37°C for 2 hours with gentle shaking. The epithelial cells were separated from the stroma and the residual limbal tissues were removed. The suspended cells were collected and plated on mitomycin-C-treated Swiss-3T3 fibroblast feeder layer in basal medium (DMEM/Ham's F12 at a ratio of 1:1, 10 mM HEPES, 0.5% DMSO) supplemented with 1 µg/ml bovine insulin, 5 ng/ml EGF and 5% FBS. Cells in passages 2 to 4 were used for the following experiments.
MTT assay
Limbal epithelial cells were seeded at 2x104 per well in 96-well culture plates. The cells were incubated overnight and were then serum starved for 24 hours. The cells were then treated with HGF or KGF in a range of 0-100 ng/ml. 24 hours after growth factor treatment, 10 µl MTT labeling reagent was added per well. 4 hours later, the cells were lysed and absorbance at 580 nm was measured. MTT assay was used to determine the optimum concentrations of HGF and KGF for limbal epithelial cell proliferation.
Measurement of limbal epithelial cell proliferation
The limbal epithelial cells were seeded into six-well culture plates at a density of 3.5x103 cells per well. On the next day, the cells were serum-starved in basal medium for 18 hours. After starvation, medium was replaced with basal medium supplemented separately with HGF (80 ng/ml) or KGF (20 ng/ml). Culture medium was replaced with the same medium every 2 days. The cell growth was monitored everyday by cell counting and photomicrographs were taken when desired.
Cell cycle analysis
The limbal epithelial cells were seeded into six-well culture plates at a density of 5x104 cells per well. On the next day, the cells were serum-starved in basal medium for 18 hours. After starvation, medium was replaced with basal medium supplemented separately with HGF (80 ng/ml) or KGF (20 ng/ml) for another 24 hours. Briefly, cells were harvested by trypsin-EDTA and pelleted at 600 g for 5 minutes. The cell pellet was resuspended in 300 µl PBS, and 700 µl ice-cold 99.9% ethanol was added to the cell suspension, mixed well and fixed at –20°C overnight. On the following day, cells were pelleted and resuspended in 1 ml PBS containing 10 g/ml propidium iodide (PI; Invitrogen, Carlsbad, CA) and 1 mg/ml RNase A (US Biological, Swampscott, MA). The cells were incubated at 37°C for 1 hour and analyzed by a FACStar (Becton–Dickinson, Mountain View, CA) with excitation at 488 nm. Approximately 10,000 cells were examined for each sample. Data were analyzed with CELLQuest software (BD Biosciences, San Jose, CA).
Limbal explant culture
Limbal tissue was procured from healthy eyes of New Zealand white rabbits (age range, 1-4 months). The animals were housed and treated according to an experimental procedure approved by the Committee for Animal Research of Chang Gung University. A 1x2 mm limbal tissue and part of the corneal stroma was separated from the limbal margin and excised from the superficial corneal stroma. The limbal explants were culture as previously described (Tsai et al., 2000). Briefly, the limbal biopsy specimen was treated with 2.4 U/ml of dispase II at 37°C for 20 minutes to loosen the epithelial layer from the underlying stroma tissue. The dispase-treated limbal biopsy was then implanted onto the denuded AM and cultured in basal medium supplemented with 1 µg/ml bovine insulin, 5 ng/ml EGF, and 5% FBS. The explant cultures were incubated in a humidified incubator (37°C, 5% CO2 in air) for 3-4 days, and explants with signs of cell migration and proliferation were selected for the subsequent experiments. The medium was replaced with the same medium and necessary supplements every 2 days. The explants were cultured for up to 2 weeks.
Measurement of epithelia outgrowth
The limbal epithelial outgrowth was visible under a phase-contrast microscope within 3-4 days. To examine the effect of HGF and KGF on cell growth, the limbal explants were serum and growth factor starved by replacing the medium with basal medium for 24 hours. The medium was then replaced with the same medium supplemented with HGF (80 ng/ml), KGF (20 ng/ml) or both. The medium was replaced every 2 days and the cultures were incubated for 10 days. The area of limbal epithelial outgrowth was measured by trypan blue staining. Briefly, the explant cultures were stained with 0.4% trypan blue and washed three times with 1x PBS. The culture dishes were photographed and the area of the outgrowth was measured with ImageQuant 5.2 (Molecular Dynamics) software.
Preparation of cell lysates and western blot analysis
For preparation of total cell lysate, the epithelial outgrowth was washed with ice-cold PBS and lysed in Mammalian Protein Extraction Reagent (M-PER; Pierce, Rockford, IL) containing 1x protease inhibitor cocktail (including AEBSF, E-64, bestatin, leupeptin, aprotinin, and sodium EDTA; Sigma). Protein concentration was determined using a Bio-Rad protein assay kit. Protein samples were fractionated on 8% SDS-polyacrylamide gel and blotted onto Immobilon (TM)-P membranes (Millipore, Bedford, MA), blocked in 5% non-fat milk in PBS-Tween, and probed with primary antibodies against Np63 (1:1000), ERK1/2-P (Thr202/Tyr204, 1:1000), JNK-P (Thr183/Tyr185, 1:1000), phosphorylated p38 MAP kinase (Thr180/Tyr182, 1:1000), Sp1 (1:1000) and GAPDH (1:10,000) for 1 hour at room temperature, followed by reaction with appropriate horseradish-peroxidase-conjugated secondary antibodies. The immunoreactive protein bands were visualized by Enhanced Chemiluminescence (ECL) (GE Healthcare, Piscataway, NJ).
RT-PCR analysis for isoform identification
For mRNA analysis, the limbal epithelial sheet outgrowth was lysed directly in the 35 mm dish by adding 1 ml TRIzol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated. Isolated RNA was reverse transcribed into cDNA using Ready-To-Go RT-PCR Beads (GE Healthcare), and the PCR reaction was performed by using GoTaq Green Master Mix (Promega, Madison, WI). PCR was performed as described (Kawasaki et al., 2006). Briefly, expression of p63 isoforms was examined by PCR with isoform-specific primer pairs modified from that of Di Iorio et al. (Di Iorio et al., 2005) (supplementary material Table S1). The PCR products were resolved on 2% agarose gels and stained with ethidium bromide.
siRNA transfection
The p63 siRNA and control non-silencing siRNA used were 5'-CCAUGAGCUGAGCCGUGAA-3' (Lee et al., 2006), and 5'-UUCUCCGAACGUGUCACGU-3', respectively (MWG-Biotech, Ebersberg, Germany). Limbal explants were cultured in basal medium supplemented with or without KGF (20 ng/ml) and were transfected with either p63 siRNA or control siRNA (80 µM) using Lipofectamine 2000 (Invitrogen). The siRNAs were replenished every 2 days. 10 days after siRNA transfection, the area of the epithelial outgrowth was stained by trypan blue and photographed. The area of the outgrowth was measured with ImageQuant 5.2 (Molecular Dynamics) software.
Construction of recombinant adenovirus vectors
Full-length human Np63 fragment was cloned as described (Chu et al., 2006). The PCR amplification product of the Np63 gene with V5 tag sequence and green fluorescence protein (GFP) were cloned into pShuttle-CMV vector (Stratagene, La Jolla, CA). The vectors were linearized with PmeI (BioLabs) and cotransformed with pAdEasy-1 skeleton vector into competent Escherichia coli cells (strain BJ5183). The successful recombinant plasmids were then linearized with PacI (BioLabs) and transfected into AD-293 cells (Stratagene) by the calcium phosphate transfection method. The AD-293 cells were incubated in a humidified incubator (37°C, 5% CO2 in air) for 10-14 days. The adenovirus vectors, designated Ad-Np63 and Ad-GFP, were propagated in AD-293 cells, collected until the appearance of cytopathic effect, and then purified by four rounds of freezing and thawing. A recombinant adenovirus carrying only GFP was used as a control.
Infection of recombinant adenovirus vectors
Limbal explants were grown in basal medium supplemented with or without KGF (5, 10, 20 ng/ml) and infected with the desired recombinant adenovirus vectors. Briefly, viral stock was diluted into 0.5 ml culture medium and added to the culture dishes. The infection reactions were incubated for 2 hours, and 1.5 ml of culture medium was then added per culture dish. The adenovirus was replenished every 2 days for a total of 10 days, and the limbal epithelial outgrowth was stained by trypan blue and photographed. The area of the outgrowth was measured as described.
Immunofluorescence staining and microscopic examination
Immunofluorescence staining was performed by standard procedures. Briefly, rabbit limbal tissues and AM-base limbal epithelial outgrowth were fixed with 4% paraformaldehyde and processed for paraffin or O.C.T. embedding. The 3 µm paraffin sections or 5 µm frozen sections were incubated with normal serum followed with the desired primary antibodies, anti-KGFR, anti-HGF receptor (HGFR), anti-p63 (1:200), anti-keratin 3 (1:200) or anti-keratin 14 (1:200). For dual-color immunostaining, samples were incubated with either cyanine 3 (Cy3)- or FITC-conjugated antibodies. Sections were mounted with anti-fade mounting medium, Gel Mount (Biomeda, Foster City, CA), and examined under a Zeiss fluorescent microscope (Oberkochen, Germany). All images were acquired using a digital photo system and processed with PhotoShop 7.0 computer software.
Statistical analysis
All the data were analyzed by Student's t-test using the SPSS statistic software, and at least three independent experiments were performed.
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Footnotes |
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This work was supported by Chang Gung Memorial Hospital Grant CMRPD170311 and National Science Council Grant NSC95-2320-B-182-029-MY3, Taiwan.
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References |
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